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研究生:洪淑貞
研究生(外文):Shu-Jen Hong
論文名稱:麴菌異黃酮氧化酵素篩選與表現系統建立
論文名稱(外文):Establishing an expression system for screening isoflavone hydroxylase in Aspergillus spp.
指導教授:戴守谷
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:光電與材料科技研究所
學門:工程學門
學類:電資工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:77
中文關鍵詞:麴菌酪胺酸酶大豆異黃酮自殺基質
外文關鍵詞:Aspergillustyrosinaseisoflavonesuicide substrate
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美白化妝品開發,主要利用之機制為抑制黑色素細胞中酪胺酸酶的活性。因此,酪胺酸酶抑制劑是目前含藥美白化妝品中廣泛使用的有效成分。然而,目前化妝品市場上所使用的有效美白成分(如對苯二酚、熊果素與麴酸)大多具有生理毒性,在使用上有其限制。因此,研究新一代酪胺酸酶抑制劑是重要的工作。異黃酮(isoflavones)屬於植物性雌激素,普遍存在於各種植物中,尤以大豆種子的含量為最高,大豆類食物中兩種主要的大豆異黃酮daidzin及genistin,可分別代謝成為不含糖基的異黃酮之衍生物daidzein與genistein。近年來證明利用米麴菌Aspergillus oryzae (A. oryzae)經過發酵程序可分別將daidzein及genistein再轉換成為異黃酮多酚7,8,4´-trihydroxyisoflavone (8-OHDe)及5,7,8,4´-tetrahydroxyisoflavone (8-OHGe)。過去研究對黃酮類化合物(flavonoids)進行了酪胺酸酶活性抑制特性分析,結果發現異黃酮多酚8-OHDe與8-OHGe屬於酪胺酸酶的自殺基質。本研究致力於建立米麴菌轉換大豆異黃酮(daidzein、genistein)形成異黃酮多酚(8-OHDe、8-OHGe)的異黃酮氧化酵素(isoflavone hydroxylase, IFH)之篩選及表現系統。米麴菌大豆異黃酮genistein轉換系統,於液態發酵轉換產生異黃酮多酚8-OHGe之時期以48小時為最佳。不同培養條件對大豆異黃酮genistein轉換能力的測試,結果顯示最適培養條件為:溫度(25 ℃) 、震盪培養轉速(200 rpm)、pH 7、鹽類濃度(10 g/L)及碳源(glucose)。不同種麴菌對大豆異黃酮genistein轉換能力的分析,結果顯示有四種麴菌包括A. flavus、A. oryzae、A. terreus及A. niger能夠轉換生成異黃酮多酚8-OHGe。本研究利用米麴菌基因體中hydroxylase、monooxygenase及cytochrome P450相關基因進行蛋白質序列相似性排比分析,篩選出四個可能具8-OHGe轉換能力之IFH相關基因,將該基因選殖到麴菌基因選殖載體pPTR-CPT,再以原生質融合轉型的方式,建立麴菌A. nidulans轉型株。雖然,轉型株中尚未發現具有大豆異黃酮genistein轉換之能力,但透過基因表現分析證實選殖的基因成功轉型進入麴菌A. nidulans。完成建立異黃酮氧化酵素IFH之篩選及表現系統之建立。因此,可供後續研究團隊利用此系統將IFH基因選殖出來。未來將透過生產重組IFH及酵素固定化來發展生物轉換系統,將可大量生產異黃酮多酚(8-OHDe、8-OHGe),也可更廣泛應用於生醫及化妝品產業。
Tyrosinase inhibitors are widely used to develop the whitening cosmetics. Several tyrosinase inhibitors such as hydroquinone, arbutin and kojic acid have been formulated in the whitening cosmetics. The agents have been found the physiological toxicity that is implicit the safety concern. Therefore, to discover the effective safety whitening agent is the important work for cosmetics industry. The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein has been investigated. The Aspergillus strains (A. flavus, A. oryzae, A. terreus and A. niger) metabolized both daidzein and genistein to 7,8,4´-trihydroxyisoflavone (8-OHDe) and 5,7,8,4´-tetrahydroxyisoflavone (8-OHGe). The 8-OHDe and 8-OHGe are potent and unique suicide substrates of mushroom tyrosinase and have highly potential in applications of the cosmetics industry. In this study we established an expression system in Aspergillus nidulans BCRC 30100 for screening the isoflavone hydroxylase, IFH from Aspergillus oryzae BCRC 32288. The enzyme could convert both daidzein and genistein into 8-OHDe and 8-OHGe. The expression system provides an approach to identify which clone is the isoflavone hydroxylase. Furthermore, we will develop a bioconversion system to produce the two suicide substrates from soy isoflavones daidzein and genistein by using immobilized recombinant IFH. The availability of the efficiency whitening agents will be extending in the applications of biomedical and cosmetics industries.
中文摘要 …………………………………………………………… i
英文摘要 …………………………………………………………… ii
誌謝 …………………………………………………………… iii
目錄 …………………………………………………………… iv
表目錄 …………………………………………………………… vi
圖目錄 …………………………………………………………… vii
一、 前言……………………………………………………… 1
1.1 研究目的………………………………………………… 1
1.2 研究背景………………………………………………… 1
1.2.1 米麴菌…………………………………………………… 1
1.2.1.1 米麴菌的分類…………………………………………… 1
1.2.1.2 米麴菌的型態…………………………………………… 2
1.2.1.3 米麴菌的應用…………………………………………… 2
1.2.1.4 利用米麴菌生產之相關酵素…………………………… 3
1.2.2 生物體內黑色素形成之機轉…………………………… 6
1.2.2.1 黑色素細胞的分佈……………………………………… 6
1.2.2.2 黑色素生成路徑………………………………………… 7
1.2.2.3 酪胺酸酶的性質………………………………………… 8
1.2.2.4 影響黑色素生成的因素………………………………… 9
1.2.3 黃酮類化合物對酪胺酸酶活性之抑制………………… 10
二、 材料與方法……………………………………………… 15
2.1 實驗流程………………………………………………… 15
2.2 菌種及基本培養基……………………………………… 15
2.3 米麴菌轉換大豆異黃酮genistein之系統建立………… 16
2.3.1 菌種的活化與保存……………………………………… 16
2.3.2 震盪培養條件…………………………………………… 16
2.3.3 8-OHGe之HPLC分析………………………………… 16
2.4 不同培養條件下米麴菌轉換大豆異黃酮genistein能力測試………………………………………………………… 17
2.4.1 溫度測試………………………………………………… 17
2.4.2 震盪培養轉速測試……………………………………… 17
2.4.3 pH值測試………………………………………………… 18
2.4.4 鹽類濃度測試…………………………………………… 18
2.4.5 碳源測試………………………………………………… 18
2.5 不同種麴菌轉換大豆異黃酮genistein能力測試……… 18
2.6 米麴菌IFH相關酵素在近緣麴菌間之蛋白質序列相似性分析及分泌特性預測…………………………………… 19
2.7 米麴菌IFH相關基因之選殖…………………………… 20
2.7.1 米麴菌之RNA萃取……………………………………… 20
2.7.2 米麴菌cDNA之製備…………………………………… 21
2.7.3 PCR引子設計…………………………………………… 21
2.7.4 進行PCR基因選殖……………………………………… 22
2.8 構築pyrithiamine resistance gene (ptrA)表現載體……… 22
2.9 建立麴菌A. nidulans轉型系統………………………… 23
2.10 米麴菌IFH相關酵素重組基因表現分析……………… 24
2.11 麴菌A. nidulans轉型株轉換大豆異黃酮genistein能力測試………………………………………………………… 25
三、 結果與討論……………………………………………… 26
3.1 米麴菌對大豆異黃酮genistein之轉換能力…………… 26
3.2 不同培養條件下米麴菌對大豆異黃酮genistein轉換能力之差異…………………………………………………… 27
3.3 不同種麴菌對大豆異黃酮genistein轉換能力之差異… 27
3.4 利用蛋白質序列相似性排比篩選具8-OHGe轉換能力之IFH相關基因…………………………………………… 28
3.5 麴菌A. nidulans外源基因表現系統之建立…………… 29
3.6 含有米麴菌IFH候選基因之重組麴菌A. nidulans之建立………………………………………………………… 31
3.7 麴菌A. nidulans轉型株對大豆異黃酮genistein轉換能力之分析…………………………………………………… 32
四、 結論……………………………………………………… 34
五、 未來展望………………………………………………… 35
參考文獻 …………………………………………………………… 36
附錄 …………………………………………………………… 71
英文論文大綱 …………………………………………………………… 75
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